Experimental factors affecting PCR-based estimates of microbial species richness and evenness

Engelbrektson, Anna; Kunin, Victor; Wrighton, Kelly; Zvenigorodsky, Natasha; Chen, Feng; Ochman, Howard; Hugenholtz, Philip

doi:10.1038/ismej.2009.153

Cited by 539 publications

(421 citation statements)

References 14 publications

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“…This same Cyanobacterialike group was only detected as a single sequence in V1-2 amplicon libraries (mouse 129S1-352). Differences in taxonomic coverage and efficiency of detection are known to occur between primer sets (Hong et al, 2009;Engelbrektson et al, 2010). In many cases, these discrepancies are not predictable based on sequence complementarity analysis (such as V4 detection of Bacteroidetes), highlighting the advantage in targeting more than one SSU rRNA gene region for analyses of taxonomic diversity (Griffen et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

Host genetic and environmental effects on mouse intestinal microbiota

et al. 2012

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The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strainspecific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.

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Section: Resultsmentioning

confidence: 99%

Host genetic and environmental effects on mouse intestinal microbiota

et al. 2012

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“…The microbiomes for three replicate samples from each invertebrate species and seawater controls were characterised. Briefly, 16S rRNA gene amplicons generated using primers 63F and 533R (Engelbrektson et al, 2010) were subjected to 454 pyrosequencing. Sequences were checked for chimeras using UCHIME ver.…”

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confidence: 99%

Coral reef invertebrate microbiomes correlate with the presence of photosymbionts

et al. 2013

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Coral reefs provide habitat for an array of marine invertebrates that host symbiotic microbiomes. Photosynthetic symbionts including Symbiodinium dinoflagellates and diatoms potentially influence the diversity of their host-associated microbiomes by releasing carbon-containing photosynthates and other organic compounds that fuel microbial metabolism. Here we used 16S ribosomal RNA (rRNA) gene amplicon pyrosequencing to characterise the microbiomes of 11 common Great Barrier Reef marine invertebrate species that host photosynthetic symbionts and five taxa in which they are absent. The presence of photosynthetic symbionts influenced the composition but not the species richness, evenness and phylogenetic diversity of invertebrateassociated microbiomes. Invertebrates without photosynthetic symbionts were dominated by Alphaproteobacteria, whereas those hosting photosynthetic symbionts were dominated by Gammaproteobacteria. Interestingly, many microbial species from photosymbiont-bearing invertebrates, including Oceanospirillales spp., Alteromonas spp., Pseudomonas spp., Halomonas spp., are implicated in the metabolism of dimethylsulfoniopropionate (DMSP). DMSP is produced in high concentrations by photosynthetic dinoflagellates and is involved in climate regulation by facilitating cloud formation. Microbiomes correlated with host taxa and replicate individuals from most sampled species grouped in distance-based redundancy analysis of retrieved 16S rRNA gene sequences. This study highlights the complex nature of invertebrate holobionts and confirms the importance of photosynthetic symbionts in structuring marine invertebrate bacterial communities.

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“…Clarke et al 2014;Liu et al 2013;Ovaskainen et al 2013;van Velzen et al 2012;Toju et al 2012). This type of error is especially acute for those studies focusing on rare taxa (Bellemain et al 2010;Engelbrektson et al 2010). For example, estimates of species richness were highly influenced by primers used: short PCR amplicons (\400 bp) produce higher number of operational taxonomic units (OTUs) than do long ones (Huber et al 2009;Engelbrektson et al 2010).…”

Section: Type I and Type Ii Errorsmentioning

confidence: 99%

“…This type of error is especially acute for those studies focusing on rare taxa (Bellemain et al 2010;Engelbrektson et al 2010). For example, estimates of species richness were highly influenced by primers used: short PCR amplicons (\400 bp) produce higher number of operational taxonomic units (OTUs) than do long ones (Huber et al 2009;Engelbrektson et al 2010). Biased PCR amplification can be caused by many factors, including universality of the primers, length of amplified noncoding regions (i.e.…”

Section: Type I and Type Ii Errorsmentioning

confidence: 99%

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Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

Zhan

MacIsaac

2014

Conserv Genet

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Identification of rare species and mapping their distributions is crucial for understanding natural species distributions and causes and consequences of accelerating species declines. However, detection of rare species in both terrestrial and especially aquatic communities typically dominated by numerous microscopic species (i.e. rare biosphere) represents a formidable technical challenge. Rapid advances in high-throughput sequencing (HTS) technologies have revolutionized biodiversity studies in the rare biosphere, and also stimulated associated debates. Here we summarize research progress, discuss debates and problems, and propose possible solutions and future studies to address these issues. In addition, we provide take-home messages for experimental design and data interpretation when utilizing HTS techniques for rare biosphere exploration in ecology and conservation biology.

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Experimental factors affecting PCR-based estimates of microbial species richness and evenness

Cited by 539 publications

References 14 publications

Host genetic and environmental effects on mouse intestinal microbiota

Host genetic and environmental effects on mouse intestinal microbiota

Coral reef invertebrate microbiomes correlate with the presence of photosymbionts

Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

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